The overall goal is to understand in molecular detail the novel RNA modification phenomenon known as uridine insertion/deletion RNA editing that occurs in the mitochondria of kinetoplastid protists. Messenger RNA transcripts of the maxicircle mitochondrial genome must be edited after transcription by insertion and deletion of U's at multiple sites to produce translatable mRNAs. This editing involves the participation of trans- acting guide RNAs which determine the precise editing sites and mediate the precise number of U's to be added or deleted. The proposal is to identify all of the proteins and RNAs involved in this process, to determine if these proteins are required for viability by RNA interference down regulation of expression, and to express the proteins in several cell systems and analyze their enzymatic or structural properties. Reconstitution of editing activities in vitro and in vivo will be performed using recombinant proteins. Structural analysis of the major core L-complex will also be performed. A functional complementation analysis of mutated L-complex proteins will also be performed in vitro and in vivo to determine the function of specific conserved motifs. The nature of the RNA mediated interactions between the L-complex and other editing complexes will be investigated. This research should yield valuable information on a novel gene regulation mechanism that is required for viability of these important human and animal parasites.